Abstract: The fecundity and survival of 6 copepod species were assessed under laboratory conditions in order to choose the best candidates to control the aquatic stages of dengue mosquitoes in the field. Females of all the 6 species (Mesocyclops aspericornis, Mesocyclops pehpeiensis, Mesocyclops woutersi, Mesocyclops thermocyclopoides, Mesocyclops ogunnus, and Megacyclops viridis) mated more than once. Multiple mating resulted in increased egg production. The reproductive ability and longevity varied among the species, and M. aspericornis had the highest values. The lowest values were observed in M. thermocyclopoides. Multiple mating of males of M. aspericornis was also observed. The paternal fecundity decreased with each additional mating. There was no difference in the paternal fecundity between the males that mated at low and high female frequencies. The sperm stored in the M. aspericornis females remained viable for 30 days after storage under moist conditions at 25 degrees C or 15 degrees C. This feature in M. aspericornis represents an additional positive factor indicating that this species is a good biological agent for controlling mosquito larvae, especially in domestic water containers that may dry intermittently.
Abstract: A field study was conducted in a village in northern Vietnam to investigate how host distribution influences Japanese encephalitis (JE) vector abundance. Indoor and outdoor collections were conducted from 50 compounds. We collected three JE vector species--Culex tritaeniorhynchus and Culex vishnui that comprised the Culex vishnui group, and Culex gelidus. Spatial autocorrelation was not observed in the mosquito assemblies at any scale larger than the house compounds. Multivariate analyses revealed that the Cx. gelidus density correlated positively with both the host proximity to the breeding sites and cattle density; however, the Cx. vishnui subgroup density correlated positively only with cattle density. These results showed that the number of cattle in a compound influenced the JE vector abundance in that compound, and the abundance of Cx. gelidus, not of the Cx. vishnui subgroup, was affected by the host proximity to the breeding sites in the village.
Abstract: The role of Anopheles funestus group in malaria transmission was investigated in Bagamoyo coastal Tanzania, in the process of characterizing the area as a malaria vaccine testing site. Mosquitoes were sampled inside houses and multiplex PCR was used to identify 649 specimens. The following species were found: A. funestus s.s. (84.3%), A. leesoni (13.6%), A. rivulorum (1.5%) and A. parensis (0.6%). Multiplex PCR of 147 blood-fed specimens showed that over half (57.1%) of the identifiable blood meals were taken from human hosts, and human blood index in A. funestus and A. leesoni was 55% and 82% respectively. Plasmodium falciparum infection rate determined by nested PCR was 11% in A. funestus s.s. Although the abundance was low, 26 specimens of A. leesoni, two of A. rivolurum and one of A. parensis were found positive for P. falciparum. The presence of four A. funestus species in Tanzania emphasizes the relevance to define precisely their spatial and temporal distribution, specific behaviour, ecology and exact role in malaria transmission.
Abstract: The mushroom genus Amanita has a spectrum of chemical compounds affecting survival and performance of animals. Ibotenic acid is one of such compounds found in some Amanita mushrooms. We studied the effects of ibotenic acid and its derivative, muscimol, on egg-to-pupa survival, pupation time, and pupal size in five Drosophila species (Diptera: Drosophilidae), Drosophila bizonata, Drosophila angularis, Drosophila brachynephros, Drosophila immigrans, and Drosophila melanogaster. The first three species are mycophagous and use a wide range of mushrooms for breeding, whereas D. immigrans and D. melanogaster are frugivorous. We reared fly larvae on artificial medium with 500, 250, 125, and 62.5 microg/ml of ibotenic acid and/or musimol. The three mycophagous species were not susceptible to ibotenic acid, whereas the two frugivorous species were affected. In experiments with D. melanogaster, muscimol was less toxic than ibotenic acid.
Abstract: Anopheline larvae generally inhabit the near-surface of aquatic habitats, but they dive and remain at the bottom of these habitats for some time. This study examined forced and voluntary diving behavior and submergence tolerance in the three major African malaria vectors, Anopheles gambiae Giles, An. arabiensis Patton, and An. funestus Giles. The former two species occur sympatrically in temporal and shallow water bodies, while the latter occurs in permanent deeper water bodies. Anopheles funestus was the most tolerant of submergence, but the larvae tended to halt their descent before reaching the bottom by attaching onto a wall. The difference in diving behavior between An. funestus and the two species in the An. gambiae complex may be an adaptation to their contrasting breeding sites, because the former species must spend considerable energy to surface in its typical breeding sites. Both An. gambiae and An. arabiensis reached the bottom and crawled along the substrate, but An. gambiae voluntarily crawled more often than An. arabiensis. The possible importance of asymmetric bottom-feeding between these two sympatric species is discussed.
Abstract: Monitoring antimalarial drug resistance is a useful epidemiologic tool and provides early detection of resistance foci. Using DNA extracted from the head/thorax of wild mosquitoes collected from Bagamoyo Coastal Tanzania, samples infected by Plasmodium falciparum (N = 89, in 2002 and N = 249 in 2004) were screened by nested polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay for mutations at Pfcrt76 and Pfmdr1-86 associated with chloroquine (CQ) resistance. The majority of isolates were of single infection (71%), and the prevalence of mutant alleles of Pfcrt76 decreased from 64.5% in 2002 to 16% in 2004; likewise, mutant Pfmdr1-86 alleles decreased from 46.6% to 2.7%. Overall, there was a decline of mutant isolates by a factor of 17 and 4 for Pfmdr1 and Pfcrt, respectively. In contrast, isolates with wild-type alleles increased significantly from < 20% in 2002 to 67.6% for Pfcrt76 and 83.5% for Pfmdr1-86 in 2004. This observation suggest a biologic trend of decrease of CQ mutants and a subsequent increase of CQ susceptible parasites in circulation after the discontinued use of CQ in 2001 as a first-line drug in Tanzania. High prevalence of susceptible P. falciparum found in circulation not only supports other reports of a decline of mutant parasites after a reduction of drug selection pressure but suggests that the fitness cost is high in mutant parasites. Typing parasite isolates from infected mosquitoes, an alternative means of data collection, has the potential to increase the spatial and temporal coverage, and this approach is practical in highly endemic regions of Africa.
Abstract: The western Kenya highland has been experiencing dramatic landuse changes in the past three decades. Landuse change has been hypothesized to be one of the mechanisms for malaria epidemics in African highlands because it can alter the physical and chemical characteristics of mosquito breeding habitats. One important landuse change in western Kenya highland is deforestation. The current study examined the effects of forestation or deforestation on the survivorship of Anopheles gambiae larvae and colonization of other aquatic insects in larval habitats in Kakamega forest (elevation 1,500-1,700 m above sea level), western Kenya. We found that the survivorship of An. gambiae larvae was reduced from 55 to 57% in habitats fully exposed to sunlight (open habitats) to 1-2% in habitats with full forest canopy coverage (forest habitats) and partial canopy coverage (forest edge habitats) in two out of three trials. The average daily water temperature of the open habitats was approximately 3-3.4 degrees C higher than the forest habitats. Insect species in the orders of Diptera, Coleoptera, and Odonata colonized the larval habitats, but the three habitat types differed greatly in the animal assemblage. Canonical correspondence analysis found that water temperature and amount of leaf litter were the significant variables associated with animal assemblages. Redundancy analysis revealed that openness and the presence of predatory animals were significantly related to An. gambiae survivorship. This result suggests that deforestation facilitates the survival of the immature stage of An. gambiae in the highland.
Abstract: The effects of the insect growth regulator pyriproxyfen were evaluated on the mortality, fecundity, longevity, and predation capability of 2 species of copepods, Mesocyclops pehpeiensis Hu and Megacyclops viridis (Jurine), under laboratory conditions. Pyriproxyfen showed no significant effects on either the development or reproduction of M. pehpeiensis at 0.1 ppm, which is a 10-fold greater concentration than the reported effective dosage for controlling mosquito larvae (0.01 ppm). In contrast, the development of M. viridis was impaired by pyriproxyfen at 0.1 ppm. An 80% reduction in nauplius survivorship was observed in the experimental (treated) group compared with the control group. Although the application of pyriproxyfen caused high mortality in the nauplius stage of this species, the pyriproxyfen-treated group developed faster, killed more mosquito larvae, yielded more eggs per oviposition event, and survived longer than the control group. These results indicate that pyriproxyfen caused mortality in the earlier stages of this sensitive species but that the surviving individuals were those that were selected for significantly faster development, better predation ability, and greater longevity during their reproductive stage. Therefore, under natural conditions, pyriproxyfen would cause modifications in the characteristics of a copepod population rather than its complete loss. Our results suggest that the combined application of copepods and pyriproxyfen to control Aedes populations is feasible. However, repeated application of pyriproxyfen may cause changes in copepod populations and communities in the affected ecosystem.
Abstract: Anopheles gambiae Giles larvae usually live near the surface of shallow and temporary aquatic habitats. How deep the larvae can dive and how long they can submerge may be related to feeding efficiency and predator avoidance. This study examined diving behavior of An. gambiae larvae in the laboratory. We recorded diving depths and larval mortality of second and fourth instars in clean water and muddy water by using deep water (32-cm) and shallow water (20-cm) columns. In deep water columns with clean water, we found that 2% of second instars and 6% of fourth instars died from diving, whereas 3% of second instars and 11% of fourth instars died in muddy water. The fourth instars dived deeper in muddy water than in clean water. The mortality rates of the fourth instars subjected to diving stimulations were significantly higher than those in the shallow water columns. Therefore, larval diving behavior may offer the benefits of predator avoidance and food acquisition but also incur energetic costs and increased mortality.
Abstract: Mae Hong Son Province in northwestern Thailand has a long history of malaria. During the last two decades the province has had one of the highest malaria incidences of all provinces in Thailand. Data were analyzed to determine whether the vector populations were stable or increasing during the last two decades and to determine the seasonal prevalence of the main vectors, and whether or not they were related to the malaria transmission peak, in the wet season. We compiled and analyzed accumulated entomological records from 1977 to 1999. The aim was to investigate long-term changes in mean densities of malaria vectors between two periods (1977-1989 and 1990-1999), and the differences in vector densities between two seasons (wet and dry). A total of 141,144 adult anophelines of 29 species were collected on indoor and outdoor human baits and animal baits during the study period. Of the main malaria vectors, the densities of Anopheles minimus s.l. and Anopheles maculatus complex increased significantly. Anopheles dirus s.l., however, was stable between the two periods. These vector populations were associated with consistently high malaria incidence in the province during the last two decades. An. minimus s.l. density was not significantly different between seasons. However, in the second period, both An. dirus s.l. and An. maculatus complex showed a tendency for higher wet season densities. This can explain the high malaria incidence in the rainy season in Mae Hong Son. Environmental and climatic factors seem to have been favorable for supporting a consistently high vector population in the province, and consequently a high malaria transmission rate during the period of study.
Abstract: Spatial distribution of mosquitoes around a bovine host was studied in November 1997 in northern Thailand (17 degrees 38'N. 99 degrees 23'E). Forty bamboo stakes were arranged 1 m apart, in 4 rays of 10, around a cow tethered in an open field. All mosquitoes found resting on the stakes were collected by aspiration between 7:00 p.m. and 11:00 p.m., sexed, and identified to species; and feeding status was categorized as fed or unfed. Collections were repeated over 8 nights, with and without the host cow. A total of 1,566 mosquitoes from 25 species (5 genera) was collected. Anopheles aconitus was the most abundant species (643 individuals), followed by An. peditaeniatus. Culex vishnui, and Cx. pseudovishnui. We found that the number of mosquitoes collected from the stakes was related to the presence of the cow host: the number of mosquitoes collected was unrelated to the compass point location of the bamboo stakes, with the exception of Mansonia uniformis; unfed mosquitoes preferred bamboo-stake resting sites that were closer to the host; the daily fed to unfed ratio of the dominant species was negatively correlated with the daily total number of mosquitoes collected; and fed and unfed mosquitoes clustered in interspecific heterogeneous groups around the host cow. Cluster analysis separated the species into 2 groups. The 1st consisted of 5 species with higher proportions of fed mosquitoes, whereas the 2nd, represented by 7 species, aggregated around the host within a distance of 1-4 m with lower proportions of fed mosquitoes. The interspecific variation in the distribution of unfed females was presumed to be due to a lack or feeding success. We discuss the significance of prebiting resting. In cases in which large numbers of mosquitoes are present, prebiting resting can be adaptive to avoid host defensive behavior triggered by attacking mosquitoes.
Abstract: Macrocyclops distinctus, Megacyclops viridis, and Mesocyclops pehpeiensis, which are common in rice fields during the summer season in Nagasaki, Japan, showed variable potentialities as biological control agents of larval Aedes albopictus, Culex tritaeniorhynchus, and Anopheles minimus in the laboratory. Macrocyclops distinctus and M. viridis, the largest copepod species, had fewer eggs within an egg clutch in nature than the smallest species, M. pehpeiensis, which also had a lower developmental time for sexual maturation (based on the appearance of the 1st clutch). Longevity as well as fecundity were influenced by nutritional conditions and varied significantly between the species. All species had shorter life spans when starved, but resistance to starvation was more pronounced in the larger species. All the species had lower clutch production when starved. Also, although the frequency of clutch production was high in M. pehpeiensis (M. pehpeiensis produced a clutch every 2 days, whereas M. distinctus and M. viridis took on average almost 3 days), total clutch production was far higher in the larger species. The copepods fed readily on mosquito larvae, with M. distinctus and M. viridis killing fewer Ae. albopictus than M. pehpeiensis, which, however, killed fewer An. minimus. These copepods exhibited a similar and limited predation against Cx. tritaeniorhynchus. Results of our study support the contention that these copepods have the potential to be used as biological control agents of immature mosquitoes. Also, our results give useful information on colony maintenance and field introduction. In particular, releasing copepods with Paramecium as food could increase their survival in the habitat of the targeted pest.
Abstract: Anopheles minimus (Theobald) is one of the most important vectors of human malaria in Southeast Asia. Morphological studies now have revealed five sibling species as its complex, designated as species A to E. The present study investigated the genetic divergence among An. minimus populations from four countries (Japan, China, Thailand and Indonesia), based on the DNA sequences data of the D3 (the third domain of the 28S ribosomal gene) and ITS2 (the second internal transcribed spacer of the ribosomal gene) is reported. The D3 and ITS2 phylogenetic trees, and the electrophoretic profile of ITS1 (the first internal transcribed spacer of the ribosomal gene) indicated that our An. minimus populations are comprised of three groups: the Japanese population as group I, the population from Guangxi Province of China (GX population) as group II, and others, as group III. The results showed the morphological similarity of group III and GX with the species complex A and B, respectively. It is interesting that both two species A (YN population) and species B (GX) occur in China, and that both species, An. minimus species A (LB-95 population) and the closer population An. flavirostris (Ludlow) (LB-00 population) appeared to be present on the Lombok Island of Indonesia, although in far separated localities. Moreover, this molecular evidence confirms the previous suggestion that the population from the Ishigaki Island of Japan should be classified as a new genetic status species E.
Abstract: Larvae of the mosquito Ae. albopictus typically develop in small aquatic sites such as tree holes and artificial containers. Organic detritus, in particular decaying leaves, is therefore their major carbon source. Here we demonstrate the importance of leaf characteristics, and in particular their rates of decay, in determining the development and survivorship of larvae. We compared the effects of a rapidly decaying leaf, the maple Acer buergerianum (Angiospermae: Aceraceae) and a slowly decaying leaf, the camphor Cinnamomum japonicum (Angiospermae: Lauraceae), on the larval development of Ae. albopictus at different larval densities in laboratory microcosms. Overall, the maple leaves provided a better substrate and the observed growth patterns could be explained on the basis of a difference in nutritive and chemical contents of the two leaf types. At the highest population density, the duration of the larval period was much shorter in maple litter microcosms. Larval mortality gradually increased with population density in the camphor treatment. In contrast in the rapidly decaying leaf litter microcosms, mortality remained low even as densities increased. Mean pupal size was greater in the individuals fed on the rapidly decaying leaf litter as well as at lower density. Size is likely to be correlated with fitness in the field. In general, rapidly decaying leaf litter will favor mosquito growth resulting in quicker development and higher population sizes. This work emphasizes the importance of the local environment on the development of vector mosquitoes and has important implications for control.
Abstract: The use of the cyclopoid copepods Macrocyclops distinctus (Richard) Megacyclops viridis (Jurine) and Mesocyclops pehpeiensis Hu (Cyclopoida: Cyclopidae) as biological control agents against the dengue vector Aedes albopictus (Skuse) (Diptera: Culicidae) was evaluated. In the laboratory their predatory ability was highest against the younger instars of Ae. albopictus and none of the three copepods killed the fourth instar. Except for M. viridis, predatory ability was affected by the size of the container: the smaller the container, the higher the predation. A 4-month field test was conducted to examine the impact of these predators on wild Ae. albopictus. Thirty artificial containers were placed in a peridomestic area to allow Ae. albopictus colonization. We showed continuous and similar oviposition responses in treated and control containers. The densities of Ae. albopictus showed considerable short-term changes and were much reduced by the copepod species. Macrocyclops and the mixture of all three provided better Ae. albopictus control than either Megacyclops or Mesocyclops alone. When larval densities peaked in the control containers in August and September, the overall reduction due to the copepods was nearly complete. Mesocyclops inoculated alone had the highest population survival. However, the growth and survival of all the copepod species was poor when the three genera were mixed. Based on their performance and survival in the trial, Macrocyclops and Mesocyclops merit consideration as bio-control agents of Ae. albopictus.
Abstract: The Anopheles minimus complex is known to comprise at least 2 sibling species (A and C) in Thailand and Vietnam. This study investigated the specific status of An. minimus on Ishigaki Island, the Ryukyu Archipelago, Japan using morphological and genetic analyses. Morphological studies revealed that almost all (99.5%) of the adult mosquitoes are characterized by the humeral pale spot on the costa of their wings, a character that partially differentiates species A and C elsewhere. A high frequency (81.4%) have a pale fringe spot at the tip of vein 1A, a character rarely observed in other An. minimus populations. Significant seasonal variation in the size of wild An. minimus mosquitoes on the island was observed, with the largest size in the winter. Scanning micrographs of the cibarial armature of females from Ishigaki Island revealed that over 90% had cone filaments clearly differing in shape from those of species A or C. The Giemsa-stained metaphase karyotypes of larval brain cells were somewhat similar to those of species A, with a few exceptions, but were very different from those reported for species C. Crossing experiments between species A (CM strain) from Thailand and the progeny of An. minimus from Ishigaki Island (ISG strain) revealed postzygotic genetic incompatibility, although no prezygotic isolation. Hybrid progeny were only obtained from CM female x ISG male. F2 hybrid progeny were not obtained, since the hybrid males were sterile or almost sterile with atrophied testes or abnormal spermatozoa, although the polytene chromosomes of hybrid larvae showed synapsis. The hybrid females backcrossed with either CM or ISG males laid eggs with significantly lowered fertility and viability. The sequence for the D3 region of the 28S gene of ribosomal DNA of the ISG strain differed from those of species A and C. In addition, sequence data from Vietnamese mosquitoes suggest that the An. minimus complex may contain additional species. The morphological, cytogenetic, molecular, and hybridization evidence together suggest the existence of another sibling species of the An. minimus complex on Ishigaki Island, which is provisionally designated An. minimus species E.
Abstract: Experiments, using the capture-mark-release-recapture technique inside large nets, were carried out in Chiang Mai, northern Thailand, to examine heterogeneity in the host preference of Japanese encephalitis (JE) vectors. A significantly higher proportion of the vector species that were initially attracted to a cow fed when released into a net with a cow than when released into a net containing a pig. However, Culex vishnui individuals that had been attracted to a pig had a higher feeding rate in a net containing a pig rather than a cow. When mosquitoes were given a choice by being released into a net containing both animals, they exhibited a tendency to feed on the host to which they had originally been attracted. This feeding preference was, however, not shown by the offspring of pig-fed individuals. We have therefore shown evidence of physiological/behavioural conditioning in the host preference of JE vectors rather than genetic variability. Our results suggest that effective control of JE might be achieved by increasing the availability of cows (the dead-end hosts of JE virus) to deflect the vectors from pigs (the amplifying host). The behavioural imprinting which we have found would tend to re-inforce the initial tendency of the vectors to bite cows.
Abstract: Anophelesflavirostris (Ludlow) from Lombok Island, Indonesia, was crossed with An. minimus species A (CM strain) from Thailand and species E (ISG strain), a new sibling species, from Japan, to determine genetic compatibility. We also compare the scanning micrographs of female cibarial armature of these three species. Both An. minimus CM and ISG females crossed with An. flavirostris males produced eggs without embryos. One An. flavirostris female crossed with an An. minimus CM male deposited unhatchable eggs with an 18% embryonation rate. The scanning micrographs of the cibarial armature clearly show significant differences among An. flavirostris and the two sibling species of the An. minimus complex. These results support the specific status of An. flavirostris. Previous records of An. minimus Theobald in this country are also discussed.
